CN106967746A - A kind of method of the Transgenic Sorghum homozygous plants of quick acquisition antiweed - Google Patents
A kind of method of the Transgenic Sorghum homozygous plants of quick acquisition antiweed Download PDFInfo
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- CN106967746A CN106967746A CN201710064529.3A CN201710064529A CN106967746A CN 106967746 A CN106967746 A CN 106967746A CN 201710064529 A CN201710064529 A CN 201710064529A CN 106967746 A CN106967746 A CN 106967746A
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- sorghum
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- antiweed
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
- C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
- C12N15/8205—Agrobacterium mediated transformation
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
- C12N15/8274—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for herbicide resistance
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/13—Plant traits
Abstract
The invention particularly discloses a kind of method of the Transgenic Sorghum homozygous plants of quick acquisition antiweed, comprise the following steps：S1. the recombinant plasmid containing target gene is imported into sorghum；S2. the IMMATURE EMBRYOS CULTURE of Transgenic Sorghum；S3. antiweed is screened；S4. Transgenic Sorghum genome, PCR detections are extracted.Because early stage of the present invention uses the Screening of Media containing herbicide, with reference to rataria increasing generation technique, it is last with for one plant in plant to enter performing PCR detection just for homologous, verify whether containing target gene, the PCR for carrying out target gene with all plant in this plant of homologous same generation again couple, which is detected, to be verified, save substantial amounts of man power and material, the detection cycle of the present invention can both realize many screenings for homozygous plants in half a year, greatly shorten detection cycle, improve genetic engineering breeding efficiency, transgenic homozygous plant is detected using the method for the present invention, its recall rate is 35% or so, with reference to the short cycle advantage of the present invention, the method of the present invention can be applied in genetic engineering breeding.
Description
Technical field
The present invention relates to Transgenic Sorghum breeding technical field, in particular it relates to a kind of turn of quick acquisition antiweed
The method of gene sorghum homozygous plants.
Background technology
Genetically modified plants (Genetically modified plants) are to possess the plant from other species genes.
Transgenic technology since the birth, due to it is with strong points, reproduction isolation between species can be broken, improve crop breeding effect
The concern of the advantages of rate, extremely breeder.Genetically modified crops bring for society continues objective economic interests, according to existing
Statistics, the world market value of transgenic seed in 2012 is 15,000,000,000 dollars, the economy that genetically modified crops in 2011 bring
Benefit is 19,700,000,000 dollars；Transgenic technology is guarantee world food safety and foodgrain self-sufficiency, sustainable development, alleviated poverty
With it is hungry, help to alleviate the challenge that climate change and global warming bring and make significant contribution.
Sorghum is not only important edible grain, or a kind of important liquor-making raw material, and China is the larger sorghum in the whole world
Producing country.In the last few years, conventional breeding was made slow progress in terms of Output of Sorghum and quality is improved, the animal nutrition such as transgenosis
New approach is opened up to solve these problems.At present, although Transgenic Sorghum is not also in China Business, Chinese scientific research personnel
Carry out disease-resistant, pest-resistant, herbicide-resistant, the research of drought-enduring and improving quality Transgenic Sorghum, and obtain preferable research
Progress.
The transgenic method of current sorghum is still based on particle gun mediated method, and the target gene that particle bombardment is imported is generally more
Copy is in the majority, and offspring's separation is serious, needs more for continuous tracing detection, could obtain the transfer-gen plant of homozygosis.Due to sorghum
Genome is larger, and the detection such as PCR is more difficult, and the workload for continuous tracing detection is very huge more, need to put into substantial amounts of people
Power and material resources.Therefore it is badly in need of a kind of method for improving transgenic breeding of invention.
The content of the invention
For the deficiency in prior art, the present invention provides a kind of Transgenic Sorghum homozygosis of quick acquisition antiweed and planted
The method of strain.
In order to solve the above-mentioned technical problem, the present invention is achieved by the following technical programs：
A kind of method of the Transgenic Sorghum homozygous plants of quick acquisition antiweed, comprises the following steps：
S1. the recombinant plasmid containing target gene is imported into sorghum；
S2. the IMMATURE EMBRYOS CULTURE of Transgenic Sorghum；
S3. antiweed is screened；
S4. Transgenic Sorghum genome, PCR detections are extracted.
Relative to prior art, beneficial effects of the present invention：
Because early stage of the present invention uses the Screening of Media containing herbicide, with reference to rataria increasing generation technique, finally just for same
Performing PCR detection is entered in source with for one plant in plant, verifies whether containing target gene, then all plants pair with this plant of homologous same generation
Strain carries out the PCR detection checkings of target gene, substantial amounts of man power and material is saved, while rataria increasing generation technique also saves purpose
Time needed for gene pure process, detection cycle of the invention is that can realize many screenings for homozygous plants in half a year, greatly
It is big to shorten detection cycle, genetic engineering breeding efficiency is improved, transgenic homozygous plant is detected using the method for the present invention, it is detected
Rate is 35% or so, with reference to the short cycle advantage of the present invention, and method of the invention can be applied in genetic engineering breeding, improves
Genetic engineering breeding efficiency.
Brief description of the drawings
Accompanying drawing herein is merged in specification and constitutes the part of this specification, shows the implementation for meeting the present invention
Example, and for explaining principle of the invention together with specification.
Fig. 1 is the schematic flow sheet of the present invention.
Fig. 2 is genome of the present invention using plasmid pAHC25.
Embodiment
The invention will be further described with the following Examples.Fig. 1 is turning for the quick acquisition antiweed of the present invention
The schematic flow sheet of the method for gene sorghum homozygous plants.
A kind of method of the Transgenic Sorghum homozygous plants of quick acquisition antiweed, comprises the following steps：
S1. the recombinant plasmid containing target gene is imported into sorghum；
S2. the IMMATURE EMBRYOS CULTURE of Transgenic Sorghum；
S3. antiweed is screened；
S4. Transgenic Sorghum genome, PCR detections are extracted.
Preferably, step S1 specific operation process described above is：
(1) target gene is inserted between Basic plasmid pAHC25 BamHI and SacI restriction enzyme restriction enzyme sites, with
Target gene replaces pAHC25 gus genes, makes target gene and antiweed bar genes in pAHC25 Basic plasmids close
It is chain；
(2) recombinant plasmid for obtaining upper step imports sorghum using agrobacterium tumefaciens-mediated transformation, obtains T0 for transgenosis
Sorghum plant；
Preferably, step S3 specific operation process described above is：
(1) take T0 for the rataria after Transgenic Sorghum plant blossom 15 days, be placed in gradient concentration 0.1mg/L, 0.5mg/L,
The medium culture of 1.0mg/L, 2.0mg/L grass fourth phosphine herbicide, culture medium is MS culture mediums, selects resistant seedling,
Continue to cultivate to 8cm or so, transplant to outdoor conventional cultivation to plant blossom；
(2) rataria after above-mentioned bloom 15 days is taken, gradient concentration 0.1mg/L, 0.5mg/L, 1.0mg/L, 2.0mg/L is placed in
The medium culture of careless fourth phosphine herbicide, culture medium is 1/2MS culture mediums, selects resistant seedling, continues to cultivate to 8cm
Left and right, is transplanted to outdoor conventional cultivation, until offspring seedling is no longer dead, the seedling is initially identified as antiweed bar genes
The Transgenic Sorghum plant of homozygosis.
Preferably, step S4 specific operation process described above is：
(1) one plant of above-mentioned Transgenic Sorghum plant for being initially identified as antiweed bar gene pures is chosen, its leaf is extracted
Piece genomic DNA；
(2) enter performing PCR to detect whether containing target gene, such as contain target gene, then the institute pair with this plant of homologous same generation
Have plant leaf DNA carry out target gene PCR detect with verifying purpose gene whether homozygosis, such as target gene homozygosis, these
Plant is Transgenic Sorghum homozygous plants.
The method of the Transgenic Sorghum homozygous plants of the quick acquisition antiweed of the present invention, carries out antiweed sieve first
Choosing, is screened simultaneously using the herbicide of various concentrations gradient, greatly shortens screening step and time, one is taken after gradient screening
Strain carries out genome extraction and PCR detects screening, reduces the workload of extraction genome and PCR detections, saves manpower financial capacity, together
When rataria increasing generation technique also save time needed for target gene homozygosis process, greatly improve the efficiency of genetic engineering breeding,
Same time, scientific research personnel can carry out multigroup operation repetitive, improve success rate of breeding.
In suitable embodiment, we make in detail for extracting its leaves genomic DNA described in above step S4
Explanation：
Preferably, its leaves genomic DNA is extracted to comprise the following steps：
S1. the centrifuge tube that 2mL is added in the appropriate sample after quartz sand and dry ice grinding is taken, is then rapidly joined
1.5mL DNA Extraction buffers and mixing of turning upside down, are mixed 3 minutes；
S2. all mixed liquors are transferred in syringe, gently pushing syringe, mixed liquor passes through sponge and miillpore filter
Filtering after, filtrate is flowed on the pellosil of adsorption column, at room temperature stand 5 minutes；
S3. new syringe is taken to be connected on adsorption column, pushing syringe, the clear liquid on pellosil can slowly pass through silicon
Glued membrane and flow in the centrifuge tube that pillar is connected below, now DNA molecular can be adsorbed on pellosil；
S4. 3 minutes are stood at room temperature after the 2mL renewed centrifuge tube, the washing lotion A for adding 500uL, and pushing syringe allows
Protein and RNA on washing lotion A eluting silica gel films；
S5. 1 minute is stood at room temperature after the 2mL renewed centrifuge tube, the washing lotion B for adding 700uL, and pushing syringe allows
The protein and RNA remained on washing lotion B eluting silica gel films；
S6. 3 minutes, pushing syringe are stood at room temperature after the 2mL renewed centrifuge tube, the eluent I for adding 500uL
Allow eluent I to elute pellosil, remove the organic solvent of residual；
S7. 1 minute is stood at room temperature after the 2mL renewed centrifuge tube, the eluent II for adding 700uL, promotes injection
Device is eluted by eluent II to pellosil, further except the organic solvent of residual；
S8. the syringe renewed, pushing syringe carries out pushing away gas drying 3 times to the silicagel column of blank pipe, then drying at room temperature 3
Minute；
S9. the 2mL renewed centrifuge tube, add 50uL storage liquid, after stand 5 minutes at room temperature, gently promote injection
It just can be collected in device, centrifuge tube and obtain high-quality DNA.
It is highly preferred that the DNA Extraction buffers composition is 5M guanidinium isothiocyanates, 10mM 2 mercapto ethanols, 50mM
Tris, 20mM EDTA, 21.3mM Triton X-100, pH 6.8；
It is highly preferred that the pellosil of the adsorption column is special silicon matrix sorbing material, it can be arranged with specific adsorption DNA
Denounce RNA and protein；
It is highly preferred that the filter layer is the sponge filter layer of large aperture and the miillpore filter layer of extra small bore diameter；The mistake
Filtering layer sponge is the material of acid-fast alkali-proof；The filter layer miillpore filter is the ceramic membrane of acid-fast alkali-proof, and aperture is the μ of 0.8nm~1
m。
The Extraction Methods of Genome that the present invention is used need not by large-scale instruments such as centrifuge, water-bath and concussion instruments,
Laboratory can be departed from, field or commodities trading market is carried to and carries out live rapid extraction genomic DNA, and extraction step letter
Just, it is time-consuming short, department's quick detections such as agricultural product monitoring and inspection and quarantining for import/export can also be can be used for, with wide application
Prospect and market value.
Experimental example
In order to verify the present invention Rapid identification antiweed Transgenic Sorghum homozygous plants method, to different cultivars
Sorghum stability, this experimental example have chosen the sorghum of three different cultivars, while with model organism arabidopsis as control,
Using the method for the present invention, the stability of the transgenic homozygous plant of different sorghum varieties, accuracy are identified.Sorghum
SbDREB2 genes are the arid response element binding proteins of sorghum, downstream are adjusted in sorghum abiotic stress a series of anti-
The expression of inverse gene, the gene is induced by abiotic stress such as arid, high salt, low temperature, ABA.
Experiment material：
Sorghum variety；Green energy 3, Xingaoliang No.2, Tiansi1 (being market purchase), arabidopsis；By Basic plasmid
PAHC25 gus reporter genes are replaced with sorghum SbDREB2 genes, build Sorghum propinquum SbDREB2 gene overexpression carriers；Will
Basic plasmid pAHC25 gus reporter genes are replaced with Arabidopsis thaliana NPR1 gene, are built Arabidopsis thaliana NPR1 gene overexpression and are carried
Body, the gene and antiweed bar gene close linkages.Each kind each handles preparation T0 for 100 plants of transfer-gen plant, warp
Antiweed preliminary screening is crossed, Genomic PCR detection is then carried out, the transgenic homozygous body plant situation such as institute of table 1 is finally obtained
Show：
Table 1
This experimental example shows, using the method for the Transgenic Sorghum homozygous plants of the Rapid identification antiweed of the present invention,
The transfer-gen plant of different sorghum varieties can be used for quickly detecting, the transgenosis of model organism arabidopsis can also be planted
Strain is used for quickly detecting, and the recall rate of its quick detection is in 35% or so, under shorter detection cycle, recall rate of the invention
The higher level of this area is reached.Above experimental example is not limitation of the present invention, and described target gene is not limited to
SbDREB2 genes and NPR1 genes, the sorghum variety are also not necessarily limited to green energy 3, Xingaoliang No.2, Tiansi1, as long as ability
Target gene is transferred to sorghum variety by the technical staff in domain under the enlightenment of the present invention, belongs to the protection category of the present invention.
Finally it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention, rather than to present invention guarantor
The limitation of scope is protected, although being explained with reference to preferred embodiment to the present invention, one of ordinary skill in the art should
Work as understanding, technical scheme can be modified or equivalent substitution, without departing from the reality of technical solution of the present invention
Matter and scope.
Claims (4)
1. a kind of method of the Transgenic Sorghum homozygous plants of quick acquisition antiweed, it is characterised in that comprise the following steps：
S1. the recombinant plasmid containing target gene is imported into sorghum；
S2. the IMMATURE EMBRYOS CULTURE of Transgenic Sorghum；
S3. antiweed is screened；
S4. Transgenic Sorghum genome, PCR detections are extracted.
2. the method for the Transgenic Sorghum homozygous plants of Rapid identification antiweed according to claim 1, its feature exists
In the step S1 specific operation process is：
(1) target gene is inserted between Basic plasmid pAHC25 BamHI and SacI restriction enzyme restriction enzyme sites, with purpose
Gene replaces pAHC25 gus genes, target gene is closely connected with the antiweed bar genes in pAHC25 Basic plasmids
Lock；
(2) recombinant plasmid for obtaining upper step imports sorghum using agrobacterium tumefaciens-mediated transformation, obtains T0 for Transgenic Sorghum
Plant.
3. the method for the Transgenic Sorghum homozygous plants of Rapid identification antiweed according to claim 2, its feature exists
In the step S3 specific operation process is：
(1) take T0 for the rataria after Transgenic Sorghum plant blossom 15 days, be placed in gradient concentration 0.1mg/L, 0.5mg/L,
The medium culture of 1.0mg/L, 2.0mg/L grass fourth phosphine herbicide, culture medium is MS culture mediums, selects resistant seedling,
Continue to cultivate to 8cm or so, transplant to outdoor conventional cultivation to plant blossom；
(2) rataria after above-mentioned bloom 15 days is taken, the careless fourth of gradient concentration 0.1mg/L, 0.5mg/L, 1.0mg/L, 2.0mg/L is placed in
The medium culture of phosphine herbicide, culture medium is 1/2MS culture mediums, selects resistant seedling, continues to cultivate left to 8cm
The right side, is transplanted to outdoor conventional cultivation, until offspring seedling is no longer dead, it is pure that the seedling is initially identified as antiweed bar genes
The Transgenic Sorghum plant of conjunction.
4. the method for the Transgenic Sorghum homozygous plants of Rapid identification antiweed according to claim 3, its feature exists
In the step S4 specific operation process is：
(1) one plant of above-mentioned Transgenic Sorghum plant for being initially identified as antiweed bar gene pures is chosen, its blade base is extracted
Because of a group DNA；
(2) enter performing PCR to detect whether containing target gene, such as contain target gene, then all plants pair with this plant of homologous same generation
Strain leaf DNA carry out target gene PCR detect with verifying purpose gene whether homozygosis, such as target gene homozygosis, these plant
As Transgenic Sorghum homozygous plants.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1359422A (en) * | 1999-04-29 | 2002-07-17 | 辛甄塔有限公司 | Herbicide resistant plants |
| CN103540608A (en) * | 2013-10-08 | 2014-01-29 | 江苏省农业科学院 | Method for rapidly obtaining transgenic wheat homozygous plant |
| CN104202968A (en) * | 2012-02-01 | 2014-12-10 | 陶氏益农公司 | Glyphosate resistant plants and associated methods |
-
2017
- 2017-02-04 CN CN201710064529.3A patent/CN106967746A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1359422A (en) * | 1999-04-29 | 2002-07-17 | 辛甄塔有限公司 | Herbicide resistant plants |
| CN104202968A (en) * | 2012-02-01 | 2014-12-10 | 陶氏益农公司 | Glyphosate resistant plants and associated methods |
| CN103540608A (en) * | 2013-10-08 | 2014-01-29 | 江苏省农业科学院 | Method for rapidly obtaining transgenic wheat homozygous plant |
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